Cellulosic ethanol: From revolutionary consolidated bioprocessing idea to proof of concept. Emile van Zyl

Transcription

1 CoER : Biofuels Department of Microbiology Faculty of Natural Sciences Cellulosic ethanol: From revolutionary consolidated bioprocessing idea to proof of concept Emile van Zyl Department Microbiology University of Stellenbosch UNIVERSITEIT STELLENBOSCH UNIVERSITY jou kennisvennoot your knowledge partner

2 Western Cape Map of Western Cape 2

3 Cape of Good Hope Arial view of Stellenbosch Cape Town - arial view of the city and Tafelberg Water Front 3

4 Stellenbosch- Jewel of the Cape Panoramic view of Stellenbosch Wine country 4

5 Stellenbosch University Central campus The Red Plane Dept. Microbiology 5

6 Content 1. Next generation technologies for cellulose conversion 2. What is Consolidated Bioprocessing? 3. Recent advances towards realizing CBP 4. Rolling out cellulosic ethanol in southern Africa 6

7 Next generation technologies for cellulose conversion 7

8 Technologies for Ethanol Production Ethanol production from sugar Yeast Alcohol recovery Sugarcane\ Sugarbeet Sweet sorghum Fermentation Distillation & dehydration Storage tank Sugar Storage tank Crashing Sugar extraction Spent yeast Fuel blending 8

9 Technologies for Ethanol Production Ethanol production from cellulosics Cellulases Yeast Alcohol recovery Pre-treatment Saccharification Fermentation Distillation & dehydration Agric Res Woody Material Grasses Water Steam explosion ~200ºC Storage tank mixing tank Chipping Grinding Spent material Fuel blending 9

10 Pretreatment for Ethanol Production Lignin Cellulose Pretreatment - produces an enzyme accessible substrate Amorphous Region Pretreatment Crystalline Region Hemicellulose Ladisch,

11 Enzymes required for CBP Cellulose Component Glucan 41.6 Xylan 15.9 Galactan 0.7 Mannan 2.2 Arabinan 0.8 Acetic acid 5 Extractives 1.4 Lignin 25.6 Ash 0.5 Total 93.7 Cellobiohydrolases Endo-glucanase β-glucosidase Endo-xylanase β-xylosidase Acetyl xylan esterase α-glucuronidase α-arabinofuransidase Endo-mannanase β-manosidase etc. Hemicellulose Partially hydrolyzed during feedstock pretreatment Glucose Xylose Manose Arabinose Galactose 11

12 Enzyme system development T. reesei secretome CBHs are the major constituent of the T. reesei cellulase system Second most important species are the EGs Broad diversity of enzymes contributes to highly active system 12

13 Estimated Cellulase Cost ($/gal EtOH) Largest Component of Recalcitrance Barrier: Cost of Cellulase d 1.00 b c f g e 0.10 a Year a) Hinman et al Appl. Biotechnol. Bioeng. 34/35: b) Hettenhaus & Glassner, 1997 ( c) NREL, Bioethanol from the corn industry. DOE/GO d) Schell, ASM Natl Meeting; McMillan, DOE/NASULGS Biomass & Solar Energy Workshops. e) Genencor & Novozyme, Press releases (e.g ). f) Petiot, Novozymes, Platts Cellulosic Ethanol & Second Generation Biofuels, g) Sheridan (Novozymes) Nature Biotech,

14 Technologies for Cellulose Conversion Biomass Processes for EtOH production Biologically- Mediated Event Cellulase Production Lignocellulose Hydrolysis Enzyme Hydrolysis Processing Strategy 1 (Each box represents a bioreactory - not to scale) O 2 SHF O 2 SSF O 2 SSCF CBP Hexose Fermentation Pentose Fermentation SHF: Separate Hydrolysis & Fermentation SSF: Simultaneous Saccharification & Fermentation SSCF: Simultaneous Saccharification & Co-Fermenation CBP: Consolidated Bioprocessing 14

15 Consolidated BioProcessing (CBP) 15

16 Consolidated BioProcessing (CBP) Fundamentals of Microbial Cellulose Utilization Microbiology and Molecular Biology Reviews 66: (2002) 16

17 Consolidated BioProcessing (CBP) Consolidated bioprocessing : update (2) Advances in Biochemical Engineering/Biotechnology 108: (2007) 17

18 Consolidated BioProcessing (CBP) Consolidated bioprocessing : update (3) Applied Microbiology and Biotechnology 87: (2010) 18

19 Technologies for Cellulose Conversion Consolidated BioProcessing (CBP) O O Glycosyl Xyl Ara Hydrolases O O O Glu Man Gal P YFG T Ethanol + CO 2 19

20 Technologies for Ethanol Production Ethanol production from cellulosics Cellulases Yeast Alcohol recovery Pre-treatment Saccharification Fermentation Distillation & dehydration Agric Res Woody Material Grasses Water Steam explosion ~200ºC Storage tank mixing tank Chipping Grinding Cooling & conditioning Spent material Fuel blending 20

21 Technologies for Ethanol Production Ethanol production from cellulosics Cellulolytic Yeast Alcohol recovery Pre-treatment Saccharification & Fermentation Distillation & dehydration Agric Res Woody Material Grasses Water Steam explosion ~200ºC Storage tank mixing tank Chipping Grinding Cooling & conditioning Spent material Fuel blending 21

22 Technologies for Cellulose Conversion Growth on amorphous cellulose (PASC) [REF] [EG1] [SFI] [CEL5] Den Haan, R., S.H. Rose, L.R. Lynd, and W.H. Van Zyl Hydrolysis and fermentation of amorphous cellulose by recombinant Saccharomyces cerevisiae. Met. Eng. 9:

23 MW (Da) 206, ,758 98,003 Expression of cellobiohydrolases in yeast CBH1 cellobiohydrolase production by yeast 54,604 37,390 29,559 20,366 7,036 Y294[CBH1] ~150X conc. Y294[CBH1] ~20X conc. Y294[CBH1] ~150X conc. treated with Pngase + control CBH1 ~250 ng, treated with Pngase Den Haan, R., J.E. Mcbride, D.C. La Grange, L.R. Lynd, and W.H. Van Zyl Functional expression of cellobiohydrolases in Saccharomyces cerevisiae towards one-step conversion of cellulose to ethanol. Enzyme Microb. Technol. 40:

24 munits/g dry cell weight Expression of cellobiohydrolases in yeast Cellobiohydrolase production by yeast % of t.c.p CBH1 CBH1-4 CBHB CBH2 CBH1 Aerobic CBH expressed in yeast CBH1 Anaerobic CBH1 requirements calculated based on ratio of CBH1 to other cellulase components in T. reesei cellulase mixtures to allow growth rate of 0.02 hr -1 Den Haan, R., J.E. Mcbride, D.C. La Grange, L.R. Lynd, and W.H. Van Zyl Functional expression of cellobiohydrolases in Saccharomyces cerevisiae towards one-step conversion of cellulose to ethanol. Enzyme Microb. Technol. 40:

25 Mascoma Corporation Technical facilities, Lebanon, NH, USA ( 25

26 Leading Investment, Unprecedented Focus on CBP Technical Focus: Overcoming the biomass recalcitrance barrier and enabling the emergence of a cellulosic biofuels industry via pioneering CBP technology integrated with advanced pretreatment Partners in Mascoma s CBP Organism Development Effort VTT, Finland Dartmouth College, USA University of Stellenbosch, ZA Three Platforms BioEnergy Science Center, USA Department of Energy, USA 1. T. saccharolyticum, thermophilic bacterium able to use non-glucose sugars 2. C. thermocellum, thermophilic cellulolytic bacterium 3. Yeast engineered to utilize cellulose and ferment glucose and xylose Multiple chances to succeed near-term & long-term 26

27 % Avicel Hydrolysis Screen CBH1 for high level expression Enzyme activity: 48 hour Avicel hydrolysis Best enzyme x13 greater than starting point mg/l secreted CBHI 30 EndoH Enzyme Production: 94 mg/l CBH1 ± 2.5% of Total cell protein in minimal medium 27

28 % Avicel Hydrolysis Screen CBH1 for high level expression Cellulose binding domain Attachment of CBD Best CBM ± 2x improvement in activity ± 2x decrease in protein level EndoH 28

29 % Avicel Hydrolysis Screen CBH2 for high level expression Enzyme activity: 48 hour Avicel hydrolysis Best enzyme x2 greater than starting point Enzyme Production: 140 mg/l CBH2 + - ± + 5% of Total cell protein in minimal medium x3 higher than EndoH starting point 29

30 % Avicel Hydrolysis Combinations of CBHs Best combination x2.5 greater than single enzyme 30

31 Protein Expression (mg/g DCW) Mascoma Cellulolytic Yeast Cellulase expression in Mascoma Yeast (robust C 5 /C 6 fermenting) Improved CBH1; ~400X Improved CBH2; ~1500X Improved host and culture conditions; ~2500X 0 CBH2 expressed (Reinikainen et al., 1992) March 2007 March 2008 Oct Cellulase expression Time-line Dec

32 Ethanol concentration, g/l Enzyme Reduction on Paper Sludge Mascoma CBP technology on 18% w/w paper sludge 60 Appearance at 120 hrs Background strain CBP + 1 mg Xylanase Background + Cellulase + BGL + Xylanase Background +BGL + Xylanase CBP strain Time (hours) 32

33 Ethanol (g/l) Enzyme Reduction on Hardwood Mascoma CBP Strain (robust C5/C6 fermenting yeast) + 22% w/w unwashed Pretreated Hardwood Equivalent performance with 2.5-fold less added enzyme Non-CBP Yeast + 1X commercial cellulases CBP Yeast + 0.4X commercial cellulases Improved CBP Yeast + 0.4X commercial cellulases Fermentation Time (hours) Further reduction likely 33

34 Rome, NY Pilot & Demonstration Plant January 2008 November

35 Acknowledgements Danie la Grange Shaunita Rose Tania de Villiers Johann Görgens Riaan den Haan Ronél van Rooyen Maryna Saayman John McBride Lee Lynd Alan Froehlich Elena Brevnova Erin Wiswall Haowen Xu Emily Stonehouse Heidi Hau Mark Mellon Kristen Deleaulte Naomi Thorngren Deidre Willies Vineet Rajgarhia Marja Ilmen Merja Penttilä 35

36 Rolling out cellulosic ethanol in southern Africa 36

37 CoER : Biofuels Department of Microbiology Faculty of Natural Sciences UNIVERSITEIT STELLENBOSCH UNIVERSITY jou kennisvennoot your knowledge partner 37

38 SBMT business model Commercialization Mascoma Exclusive license (>30 patents, process designs) Value addition to process Thank you! SBMT SU Africa demonstration SBMT holds the exclusive rights to Mascoma technology for Southern Africa 38

39 ZAR (million) log scale Costs vs timeline for technology development 2 Demonstration Plant 3 Paper Sludge? Woodchips commercial ~R M 4 Integrated 1 st /2 nd generation (sugar cane) >R1 B Mobile conversion unit R5-10 M R M Years 39

40 CoER : Biofuels Department of Microbiology Faculty of Natural Sciences Thank you UNIVERSITEIT STELLENBOSCH UNIVERSITY jou kennisvennoot your knowledge partner